The present invention relates to methods for the determination of target nucleotide sequences, and to diagnostic kits suitable for such methods.
A group of inventors, including some of the present inventors, have disclosed in U.S Ser. No. 607,885, and in several further patent applications discussed below, a nucleic acid assay based upon strand displacement. Such assay employs a reagent complex of:
(a) a probe polynucleotide (P) having a target binding region (TBR) complementary to the target nucleotide sequence (TNS) to be detected, and PA1 (b) a labeled polynucleotide (L) (or signal strand) bound in the reagent complex to the probe polynucleotide (P) in at least a portion of the target binding region (TBR).
The portion of the labeled polynucleotide (L) so-bound is sometimes referred to in those applications as the pairing segment (PS). In the assay, the target nucleotide sequence (TNS) binds to the target binding region (TBR) and displaces the labeled polynucleotide (L). The displaced labeled polynucleotide is then detected as a measure of the presence and amount of target nucleotide sequence (TNS) in the sample.
In those patent applications, separation of displaced labeled polynucleotides from intact reagent complexes is described for many forms of the invention. Two forms of separation involve: (a) a reagent complex in which the probe polynucleotide is immobilized and (b) a reagent complex in which the probe polynucleotide is in solution with a pendant affinity moiety (e.g., biotin) for post-displacement immobilization. In U.S Ser. No. 729,501 of Unger, et al. various other groups in or of the probe (e.g., a poly-dC tail) are disclosed for post-displacement immobilization. In such cases of immobilized probes or post-displacement immobilization of the probe, the displaced labeled polynucleotides remain in solution for detection. In the usual event, only a small proportion (1% to 0.00001%) of labeled polynucleotides are displaced, however,a variety of mechanisms such as strand breakage of regent complexes detachment of immobilized reagent complexes from the solid or inefficient trapping of immobilizable complexes, can result in labeled polynucleotides remaining in solution in the absence of specific displacement events. These mechanisms are a source of background for the assay. The level of tolerable background will depend upon both the number of reagent complexes used in the reaction and the amount of analyte to be detected. For example, to detect 10.sup.6 analytes in a reaction with 10.sup.11 reagent complexes, 0.001% (10.sup.6) or fewer complexes must be released or not immobilized as background for any signal to be detected with a (signal & background)/background ratio of at least 2/1.
In U.S Ser. No. 607,885, embodiments are shown (e.g., in FIG. 2A) wherein the labeled polynucleotide L bears an affinity moiety (biotin) for post-displacement immobilization. While such technique concentrates the signal (a desirable result in many cases), it is unlikely to aid in background reduction, since displaced labeled polynucleotides and detached reagent complexes each have biotin and are likely to be immobilized with substantially equal efficiency.
Other pending U.S. patent applications concerned with strand displacement of nucleic acids include the following:
______________________________________ 684,305 December 10, 1984 Collins, et al. 684,308 December 10, 1984 Williams, et al. 729,501 May 2, 1985 Unger, et al. 729,503 May 2, 1985 Vary, et al. 729,504 May 2, 1985 Fritsch, et al. 777,796 September 19, 1985 Fritsch, et al. ______________________________________
In addition; U.S Ser. No. 790,671 of Ellwood, et al, discussed more fully below, relates to an assay involving hybridization, but not strand displacement.